RESEARCH ARTICLE

Rapid Karyotype Evolution in Lasiopodomys

Involved at Least Two Autosome – Sex

Chromosome Translocations

Olga L. Gladkikh1☯, Svetlana A. Romanenko1,2☯*, Natalya A. Lemskaya1, Natalya

A. Serdyukova1, Patricia C. M. O’Brien3, Julia M. Kovalskaya4, Antonina

V. Smorkatcheva5, Feodor N. Golenishchev6, Polina L. Perelman1,2, Vladimir

A. Trifonov1,2, Malcolm A. Ferguson-Smith3, Fengtang Yang7, Alexander

S. Graphodatsky1,2

1 Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences,

Novosibirsk, Russia, 2 Novosibirsk State University, Novosibirsk, Russia, 3 Cambridge Resource Centre for

Comparative Genomics, Department of Veterinary Medicine, University of Cambridge, Cambridge, United

Kingdom, 4 Severtzov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia,

5 Department of Vertebrate Zoology, Saint Petersburg State University, Saint Petersburg, Russia,

6 Zoological Institute, Russian Academy of Sciences, Saint Petersburg, Russia, 7 Wellcome Trust Sanger

Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom

☯ These authors contributed equally to this work.

* rosa@mcb.nsc.ru

Abstract

The generic status of Lasiopodomys and its division into subgenera Lasiopodomys (L. man-

darinus, L. brandtii) and Stenocranius (L. gregalis, L. raddei) are not generally accepted

because of contradictions between the morphological and molecular data. To obtain cyto-

genetic evidence for the Lasiopodomys genus and its subgenera and to test the autosome

to sex chromosome translocation hypothesis of sex chromosome complex origin in L. man-

darinus proposed previously, we hybridized chromosome painting probes from the field vole

(Microtus agrestis, MAG) and the Arctic lemming (Dicrostonyx torquatus, DTO) onto the

metaphases of a female Mandarin vole (L. mandarinus, 2n = 47) and a male Brandt’s vole

(L. brandtii, 2n = 34). In addition, we hybridized Arctic lemming painting probes onto chromo-

somes of a female narrow-headed vole (L. gregalis, 2n = 36). Cross-species painting

revealed three cytogenetic signatures (MAG12/18, 17a/19, and 22/24) that could validate

the genus Lasiopodomys and indicate the evolutionary affinity of L. gregalis to the genus.

Moreover, all three species retained the associations MAG1bc/17b and 2/8a detected previ-

ously in karyotypes of all arvicolins studied. The associations MAG2a/8a/19b, 8b/21, 9b/23,

11/13b, 12b/18, 17a/19a, and 5 fissions of ancestral segments appear to be characteristic

for the subgenus Lasiopodomys. We also validated the autosome to sex chromosome

translocation hypothesis on the origin of complex sex chromosomes in L. mandarinus. Two

translocations of autosomes onto the ancestral X chromosome in L. mandarinus led to a

complex of neo-X1, neo-X2, and neo-X3 elements. Our results demonstrate that genus

Lasiopodomys represents a striking example of rapid chromosome evolution involving both

autosomes and sex chromosomes. Multiple reshuffling events including Robertsonian

PLOS ONE | DOI:10.1371/journal.pone.0167653 December 9, 2016 1 / 15

a11111

OPENACCESS

Citation: Gladkikh OL, Romanenko SA, Lemskaya

NA, Serdyukova NA, O’Brien PCM, Kovalskaya JM,

et al. (2016) Rapid Karyotype Evolution in

Lasiopodomys Involved at Least Two Autosome –

Sex Chromosome Translocations. PLoS ONE 11

(12): e0167653. doi:10.1371/journal.

pone.0167653

Editor: Igor V. Sharakhov, Virginia Tech, UNITED

STATES

Received: September 20, 2016

Accepted: November 17, 2016

Published: December 9, 2016

Copyright: © 2016 Gladkikh et al. This is an open

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the paper and its Supporting Information

files.

Funding: The study was funded by the by the

Russian Science Foundation (RSF, http://rscf.ru/)

under project No. 16-14-10009 to ASG. This work

was partially supported by the Programs of the

Federal Agency for Scientific Organizations (FASO

Russia, http://fano.gov.ru/) No. 01201351185 to

FNG for the animals collection and No. 0310-2016-

0002 for cell culture collection to ASG. Significant

fusions, chromosomal fissions, inversions and heterochromatin expansion have led to the

formation of modern species karyotypes in a very short time, about 2.4 MY.

Introduction

Rodentia is the most species-rich mammalian order and includes several important laboratory

model species. The tribe Arvicolini is particularly notable for its extensive phenotypic and

chromosomal variations which raised many questions about species dispersal and genomic

processes accompanying speciation.

The generic status of Lasiopodomys Lataste, 1887 from the subfamily Arvicolinae (Criceti-

dae, Rodentia) is not universally accepted because of contradictions between the morphologi-

cal and molecular data. Until recently, the genus Lasiopodomys included L. mandarinus Milne-

Edwards, 1871, L. brandtii Radde, 1861, and L. fuscus Buchner, 1889 [1]. However, molecular

data have indicated a kinship between Lasiopodomys and Stenocranius [2–5], while L. fuscushas been considered part of the large clade of East Asian voles of genus Neodon Hodgson, 1849

[6]. Based on these findings Pavlinov and Lissovsky [7] subdivided Lasiopodomys into two sub-

genera with three species: subgenus Lasiopodomys (L. mandarinus and L. brandtii) and subge-

nus Stenocranius (L. gregalis Pallas, 1779). Recently, it has been shown that L. gregalis is

represented by two species–L. gregalis and L. raddei Poljakov, 1881 [8,9]. According to the

analysis of nuclear genes the genus Lasiopodomys originated within the tribe Arvicolini Gray,

1821, approximately 2.4 million years ago (MYA), and the division of that genus into subge-

nera Stenocranius and Lasiopodomys occurred about 1.8 MYA [3,10].

Here we aim to test the validity of the subgeneric division of Lasiopodomys through cross-

species chromosome painting and to define cytogenetic signatures (fusions, fissions, chromo-

somal association) for it as well as for separation of the Lasiopodomys genus from other arvico-

lins. Previously we described the karyotype of L. gregalis from the eastern border of the species

range and established its chromosomal homology with painting probes of the field vole (Micro-tus agrestis Linnaeus, 1761) [11], but other Lasiopodomys species have not been studied by

comparative chromosome painting.

The Lasiopodomys species are notable for their high karyotype variation. Two species, L.

brandtii and L. gregalis, have stable diploid chromosome numbers of 34 and 36, respectively,

and constant chromosome morphology, in spite of significant morphological variations in L.

gregalis from different populations [3,10,12–15]. The diploid chromosome number of L. man-darinus varies between 47 and 52 in different populations: 2n = 47–48, NF = 53–55 in Mongo-

lia and Buryatia [16] and 2n = 47–52, NF = 53–55 in China [17–19]. Besides the variation in

diploid chromosome number, polymorphism in chromosome morphology often involving

two pairs of autosomes (№1 and №2) and the sex chromosomes was described for L. mandar-inus [17–23]. Moreover, L. mandarinus is one of the few rodent species documented to have

an unusual sex chromosome system [17–23]. Three types of sex chromosome systems (XX,

XO, XY) with heteromorphic X chromosomes were described for the species based on C- and

G-banding comparisons [23].

The current hypothesis on the origin of the unusual sex chromosomes morphology in L.

mandarinus, which suggests that it is the result of translocation of autosomes onto sex chromo-

somes, was introduced by Wang et al. [17]. This hypothesis received support from the analysis

of synaptonemal complexes in the Mandarin vole, establishing that the sex chromosomes of L.

mandarinus (XY) pair and recombine at pachytene. It should be noted that the sex chromo-

somes of L. gregalis and L. brandtii do not synapse or recombine during meiosis [24]. We set

Karyotype Evolution in Lasiopodomys

PLOS ONE | DOI:10.1371/journal.pone.0167653 December 9, 2016 2 / 15

funding was also provided by the Russian

Foundation for Basic Research (RFBR, http://www.

rfbr.ru/) grants No. 14-04-00451 to SAR, No. 15-

04-00962 and No. 15-29-02384 both to ASG, No.

16-04-00983-a to FNG for the animals and tissue

cell collections.

Competing Interests: The authors have declared

that no competing interests exist.

Abbreviations: 2n, diploid number of

chromosomes; AAK, ancestral Arvicolinae

karyotype; AMiK, ancestral karyotype of the genus

Microtus; DTO, Dicrostonyx torquatus; FISH,

fluorescence in situ hybridization; GTG-banding, G-

banding by trypsin using Giemsa; ITS, interstitial

telomeric sequences; LAK, ancestral karyotype of

the genus Lasiopodomys; LBRA, Lasiopodomys

brandtii; LGRE, Lasiopodomys gregalis; LMAN,

Lasiopodomys mandarinus; MAG, Microtus

agrestis; MYA, million years ago; MY, million years;

NFa, fundamental autosomal number; sLAK,

ancestral karyotype of the subgenus

Lasiopodomys.

out to test the autosome to sex chromosome translocation hypothesis of the complex sex chro-

mosome origin in L. mandarinus through the use of molecular cytogenetic tools. We also used

two sets of painting probes, from the field vole (Microtus agrestis) and the Arctic lemming

(Dicrostonyx torquatus Pallas, 1778) [25,26], to track evolutionary chromosome rearrange-

ments in karyotypes of the species L. mandarinus (LMAN), L. brandtii (LBRA), and L. gregalis(LGRE).

Materials and Methods

Ethics statement

All experiments were done in accordance with the European Community Council Directive of

22 September 2010 (2010/63/EU) and approved by the Committee on the Ethics of Animal

Experiments of the Institute of Molecular and Cellular Biology (IMCB) SB RAS, Russia, and

the Committee on the BioEthics of the Zoological Institute, RAS, Saint Petersburg, Russia.

Species sampled

One female L. mandarinus, one male L. brandtii, and one female L. gregalis specimens used in

this study were obtained from the laboratory colonies housed at the Leningrad Zoo and the

Institute of Zoology of Russian Academy of Sciences (L. mandarinus originated from Selen-

ginsky District, Buryatia; L. brandtii–from Cape Telly, Torey Lake, Borzinskiy District, Chita

region, Zabaikalsky Kray; L. gregalis–from River Kadala, Chitinsky District, Zabaikalsky Kray).

The voles were sacrificed by cervical dislocation. Fibroblast cell lines from skin, lung, and

tail biopsies and chromosome suspensions were obtained in the Laboratory of Animal Cytoge-

netics, the IMCB SB RAS, Russia. Primary fibroblast cell lines were established using enzy-

matic treatment of tissues as described previously [27,28]. All cell lines were deposited in the

IMCB SB RAS cell bank (“The general collection of cell cultures”, № 0310-2016-0002).

Chromosome preparation and chromosome staining

Metaphase chromosome spreads were prepared from chromosome suspensions obtained from

early passages of primary fibroblast cultures as described previously [29–31].

C-banding followed the method of [32] with some modifications [33]. Briefly, the slides

were treated with 0.2 N HCl for 20 minutes at room temperature, rinsed in distilled water, and

incubated in a freshly prepared 4.6% solution of Ba(OH)2 at 53˚C for 2–4 min. After thorough

rinsing in 0.2 N HCl and several changes of distilled water, the slides were incubated for 1 h at

60˚C in 2X SSC, rinsed briefly with distilled water and stained with 2% Giemsa for 7 min.

G-banding was performed on chromosomes of all three species prior to FISH using the

standard trypsin/Giemsa treatment procedure [34].

Fluorescence in situ hybridization (FISH)

The field vole and Arctic lemming painting probes were generated in the Cambridge Resource

Centre for Comparative Genomics by DOP-PCR amplification of flow sorted chromosomes

and labeled with either biotin or digoxigenin by DOP-PCR amplification as described previ-

ously [25,26,35,36]. The telomeric DNA probe was generated by PCR using the oligonucleo-

tides (TTAGGG)5 and (CCCTAA)5 [37]. Clones of human ribosomal DNA containing the

complete 18S-rRNA and 28S-rRNA genes were obtained as described in [38]. FISH was per-

formed following previously published protocols [30,31]. Images were captured using VideoT-

est-FISH software (Imicrotec) with a JenOptic CCD camera mounted on an Olympus BX53

microscope. Hybridization signals were assigned to specific chromosome regions defined by

Karyotype Evolution in Lasiopodomys

PLOS ONE | DOI:10.1371/journal.pone.0167653 December 9, 2016 3 / 15

G-banding patterns previously photographed and captured by the CCD camera. All images

were processed using Corel Paint Shop Pro X2 (Jasc Software).

Results

The male of L. brandtii and female of L. gregalis have 2n = 34, NF = 67 and 2n = 36, NF = 54,

respectively. The investigated female of L. mandarinus has 2n = 47 and NF = 55 with 22 pair of

autosomes, one metacentric neo-X1 chromosome, one submetacentric neo-X2 chromosome

and one small acrocentric neo-X3 (Fig 1A). The metacentric neo-X1 chromosome has three

large C-positive blocks on the q-arm (Fig 2A).

The field vole and the Arctic lemming painting probes were used to establish chromosomal

homologies in the Mandarin vole, Brandt’s vole, and narrow-headed vole (Fig 1). The 24 M.

agrestis autosomal chromosome probes revealed 39 and 34 conserved segments in L. mandari-nus and L. brandtii karyotypes, respectively (Fig 1A and 1B and S1 Table). In the L. mandari-nus karyotype the MAGX (Microtus agrestis X chromosome) probe hybridizes to the p-arm of

the metacentric neo-X1 chromosomes and to the interstitial part of the q-arm of the submeta-

centric neo-X2 chromosome. MAG13 paints parts of the neo-X1 and neo-X2 chromosomes,

while MAG17 and MAG19 probes hybridize to the distal q-arm of neo-X2 and paint the whole

neo-X3 of L. mandarinus (Fig 1A).

The 23 D. torquatus probes reveal 42, 36 and 32 conserved segments in L. mandarinus, L.

brandtii and L. gregalis karyotypes, respectively (Fig 1A and 1C and S1 Table). In the L. man-darinus karyotype the DTOX1 probe hybridizes to the p-arm of the metacentric neo-X1 chro-

mosomes and a middle part of the q-arm of the submetacentric neo-X2 chromosome (Fig 1A).

Four probes of DTO (2, 12, 13, and 19) hybridize to the heteromorphic pair of sex chromo-

somes (Fig 1A). Examples of fluorescence in situ hybridizations are shown in Fig 3.

The telomeric DNA probe was localized onto chromosomes of all studied species (Fig 3E–

3G). Interstitial Telomeric Sequences (ITS) were detected in L. mandarinus and L. brandtii(Fig 1A and 1C). In the L. brandtii karyotype telomeric signals are much weaker than pericen-

tromeric ones distributed on chromosomes 2, 4, 5, 7, 10, 11, 13, 14, 15, and 16 (Fig 3F). In L.

mandarinus non-centromeric ITSs were localized on chromosome 1 and on the neo-X2 chro-

mosome (Fig 3E).

We detected six rDNA clusters in L. brandtii (on chromosomes 4, 11, 12, 14, 15, and 16)

and L. gregalis (on chromosomes 9, 11, 12, 13, 15, and 16) and three rDNA clusters in L. man-darinus (on chromosomes 3, 18, and 22) (Fig 3E–3G). In L. brandtii and L. gregalis, the ribo-

somal DNA was located at chromosomal segments homologous to MAG1/17 and MAG21 (in

associations MAG1/17/12/18 (LBRA4), MAG8/21 (LBRA12) in L. brandtii and MAG1/17

(LGRE12), MAG20/23/21 (LGRE9) in L. gregalis).

Discussion

Unclear taxonomic status of the Lasiopodomys and its species composition as well as the

unusual structure of sex chromosomes described in L. mandarinus make this rodent genus

especially interesting for cytogenetic studies. In spite of being actively involved in molecular

phylogenetic investigations, voles have remained out of deep chromosomal studies for a long

time. Here we used cytogenetic tools to test the validity of the subgeneric division of Lasiopod-omys and its phylogenetic relationships to other Arvicolinae. The use of cross-species chromo-

some painting allowed us to track evolutionary chromosome rearrangements in karyotypes of

three Lasiopodomys species, and to support not only the autosome to sex chromosome translo-

cation hypothesis in L. mandarinus but also to describe the sex chromosomal system in detail.

Karyotype Evolution in Lasiopodomys

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Karyotype Evolution in Lasiopodomys

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The complex sex chromosome system in L. mandarinus

A particular combination of sex chromosomes usually determines whether an individual is

male or female. Most mammalian species have a conventional XX/XY sex chromosome system

with the Y-borne testis determining SRY gene. It was proposed that sex chromosomes origi-

nated from a pair of autosomes where one of the homologs acquired the sex determining locus

[39,40]. Further evolutionary processes led to the accumulation of sex-specific genes on both X

and Y chromosomes, suppression of recombination between them and rapid degeneration of

Y, occasionally resulting in a complete loss of the Y-chromosome [39]. This process can be fur-

ther complicated by interchromosomal rearrangements (fusions, fissions, translocations),

which result in the emergence of either neo-sex chromosomes or multiple sex chromosome

complexes. Neo-sex chromosomes originate from the addition of autosomes or autosomal seg-

ments onto original sex chromosomes [41] and in some cases subsequent fission or new

fusions of such neo-sex chromosomes could lead to emergence of multiple sex chromosomes

[42]. Autosomal material is often added to one of the sex chromosomes or to both of them

[36,43,44]. Even in cases with a known composition of the neo-sex chromosome complexes,

many questions remain unanswered regarding behavior of those elements in meiosis, localiza-

tion of sex-determining loci and the role of neo-chromosome complexes in speciation etc.

Although some sex chromosome systems other than XX/XY are found within various

mammalian taxa, rodents demonstrate the widest range of sex chromosome systems and even

mechanisms of sex determination (see [45] and references therein). Many species with unusual

sex chromosome systems belong to the Arvicolinae subfamily, and the Mandarin vole is one of

them. Polymorphism in the sex chromosomes of L. mandarinus has been described previously

based on G- and C-banding analysis only [16–19,22]. Wang et al. proposed that such a poly-

morphism might be caused by the translocation of autosomes to the X chromosome [17].

Fig 1. GTG-banded karyotypes of studied species. a–L. mandarinus, b–L. brandtii, c–L. gregalis. Black dots mark the position of

centromeres. Vertical black bars mark the localization of M. agrestis (MAG) chromosome painting probes, while vertical grey bars

mark the localization of D. torquatus (DTO) painting probes. Numbers along the vertical lines correspond to chromosome numbers

of M. agrestis and D. torquatus. Black triangles indicate sites of localization of rDNA clusters; grey triangles indicate localization of

the largest interstitial telomeric blocks.

doi:10.1371/journal.pone.0167653.g001

Fig 2. C-banding. a–L. mandarinus, b–L. brandtii, с –L. gregalis. Scale bar is 10 μm.

doi:10.1371/journal.pone.0167653.g002

Karyotype Evolution in Lasiopodomys

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Here the application of chromosome painting provides evidence for the origin of neo-sex

chromosomes in L. mandarinus by at least two independent autosome-sex chromosome trans-

location events. The complex of sex chromosomes in the L. mandarinus female described here

consists of one metacentric chromosome (neo-X1), one submetacentric chromosome (neo-X2)

and one small acrocentric referred to here as “neo-X3”. At least two pairs of ancestral auto-

somes participated in the formation of these neo-X chromosomes. We propose the following

description of the karyotype of the female Mandarin vole: 47, neo-X1, neo-X2, neo-X3, where

“47” is the diploid number and “neo-X” is a description of sex chromosomes.

This finding is consistent with the theory proposed based on the unusual synapsis in meio-

sis of the Mandarin vole [24]. The XY bivalent of L. mandarinus contains two pairing regions.

Both pairing regions are relatively long and both take part in regular recombination, similar to

the human pseudoautosomal regions [24]. These regions may have originated by de novotranslocation of autosomal segments, as proposed for the human pairing regions [46].

The formation of the metacentric neo-X1 was accompanied by heterochromatin accumula-

tion in the part of the X chromosome homologous to MAG13. It seems likely that either the

ancestral X chromosome or the ancestral autosomal segment carried a heterochromatic block

that prevented the spread of chromosome inactivation into the autosomal compartment after

the translocation [47]. We conjecture that a fusion of MAG13/X with MAG17/19 took place

relatively recently as the region of fusion retained ITS sites (Figs 1A and 3E–3G). Thus we

Fig 3. Examples of fluorescence in situ hybridization. a–MAGX (green) and MAG13-14 (red) onto L. mandarinus chromosomes, b–DTO10-12 (green)

and DTO2 (red) onto L. mandarinus chromosomes, c–DTO13 (green) and DTO9 (red) onto L. gregalis chromosomes, d–DTO2 (green) and DTO19 (red) onto

L. brandtii chromosomes. Examples of fluorescence in situ hybridization of the 18S/28S-rDNA probe (green) and telomeric DNA probe (red): e–L. mandarinus

(white arrows indicate localization of the largest interstitial telomeric blocks), f–L. brandtii, g–L. gregalis. Scale bar is 10 μm.

doi:10.1371/journal.pone.0167653.g003

Karyotype Evolution in Lasiopodomys

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have characterized the complex chromosomal composition of the sex trivalent in the female

Mandarin vole. Nevertheless, to understand this phenomenon at the molecular and sequence

level further studies are required, involving additional individuals from both sexes and an in-

depth study of the localization and function of sex-determining genes.

Cytogenetic data support a close relationship between subgenus

Lasiopodomys and subgenus Stenocranius

Recently there has been much discussion on taxonomic revision of the genus Lasiopodomysconcerning the status of the whole taxon, its species composition, and in particular the division

of the genus into subgenera Stenocranius (L. gregalis and L. raddei) and Lasiopodomys (L. man-darinus and L. brandtii) [2–5,8,9]. Often, especially when morphologically similar species are

studied, cytogenetic characters provide additional phylogenetic data for possible problem-

solving.

The presence of MAG1bc/17b (an association common for almost all Arvicolinae and con-

taining the fragment MAG1b/17b which was also found in some Cricetinae species) and

MAG2/8a (common for all Arvicolinae) in karyotypes of the three studied Lasiopodomys spe-

cies may be considered as a synapomorphic trait of the Arvicolinae subfamily. MAG12/18,

17a/19, and 22/24 associations have not been found previously in karyotypes of other rodents

and may be the defining markers for the genus Lasiopodomys (Table 1). It is important to stress

that L. brandtii and L. gregalis carry rDNA clusters at chromosomal segments homologous to

MAG1bc/17b, probably sharing the same origin (the same for rDNA clusters in L. brandtiiand L. gregalis located at chromosomal segments homologous to MAG21).

The associations MAG2a/8a/19b, 8b/21, 9b/23, 11/13b, 12b/18, and 17a/19a were found in

the karyotypes of L. mandarinus and L. brandtii and could be characteristic for the subgenus

Lasiopodomys. Although some of these associations seem to have a shared origin with other

Table 1. Distribution of shared syntenic segment associations.

Association of MAG

chromosomes

Association of DTO

chromosomes

LMAN LBRA LGRE Presence of association in other taxonomic

groups

Reference

1b/17b 5 + + + common for all Arvicolinae and some Cricetinae

species

[25]

1bc/17b 4/5 + + + all Arvicolinae except Dicrostonyx torquatus and

Ellobius talpinus

[25]

2/8a 1 + + + all Arvicolinae

8/19 3/13 Microtus dogramacii; Alexandromys maximowiczii [11]

8/19 1/13 + +

2a/8a/19b 1/13 + +

8b/21 3/15 + +

9b/23 5/2 + + Ellobius talpinus [48]

9/23 18/2 Mesocricetus auratus [25]

11/13b 3/12a + + Arvicola amphibius [25]

11/13 3/12 Arvicola amphibius [25]

12b/18 Y1qa/X2 + + +

12/18 Y1q/X2 +

17a/19a 2/19/13a + + +

17a/19 2/19/13b +

22/24 17/20/21 + +

MAG–M. agrestis, DTO–D. torquatus, LMAN–L. mandarinus, LBRA–L. brandtii, LGRE–L. gregalis.

doi:10.1371/journal.pone.0167653.t001

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arvicolin or cricetin species, the use of Arctic lemming painting probes turned out to be instru-

mental in resolving and disproving these seemingly shared associations (Table 1).

Despite the fact that MAG12/18 and MAG17a/19 are the key associations synapomorphic

for the whole genus Lasiopodomys, the fissions of MAG12 and MAG19 leading to the forma-

tion of MAG12a, MAG12b/18 and MAG17a/19a, MAG19b (included in MAG2a/8a/19b),

respectively, are characteristic only for the subgenus Lasiopodomys. The karyotype of L. man-darinus is characterized by a high fragmentation in comparison with the karyotypes of its sister

species. The absence of the association MAG22/24 (= DTO17/20/21) in the L. mandarinus kar-

yotype was probably a secondary event due to chromosome fission. However, we cannot

exclude the possibility that the presence of the association in karyotypes of L. brandtii and L.

gregalis only was a result of convergent evolution. In this case L. mandarinus retained the

ancestral condition. Thus, the origin of the association MAG22/24 has not been unambigu-

ously defined.

The shared chromosomal associations found here seem to be in agreement with the evolu-

tionary closeness of L. mandarinus and L. brandtii and their isolation from L. gregalis, provid-

ing cytogenetic evidence for the taxonomic division of subgenera based on DNA sequence

analyses [2–5]. Moreover, the cytogenetic data reveal possible chromosomal signatures (12/18,

17a/19, and 22/24) that support the separate position of the genus inside Arvicolinae.

Rapid karyotype evolution in Lasiopodomys

The number of chromosomal rearrangements occurring in a certain evolutionary period indi-

cates the rate of evolutionary karyotype reorganization. Previously it was revealed that the rate

of karyotype evolution varies greatly (in several folds) across the mammalian phylogenetic tree

[49]. In rodents the average rate of karyotype evolution has been determined as one fusion/fis-

sion per MY [50]. Arvicolinae belongs to the group of species characterized by an even higher

tempo of chromosomal reorganization [11].

The cladistic analysis of chromosomal rearrangements did not provide a well-resolved tree

with valid support for arvicolin genera branching due to the shortage of phylogenetically infor-

mative chromosomal characters (our unpublished data). However, the availability of the

molecular phylogeny of Arvicolinae species [3], a previously proposed ancestral karyotype for

the subfamily Arvicolinae (Ancestral Arvicolinae Karyotype, AAK) [25], an ancestral karyo-

type for the tribe Arvicolini (AMiK) [11], and cytogenetic data on Lasiopodomys species-spe-

cific characters allow us to track chromosomal exchanges and calculate the tempo using

molecular estimates of divergence times (Fig 4).

Most phylogenetic studies support the early branching of the common ancestor of the

genus Lasiopodomys from the stem lineage of tribe Arvicolini [3,7,10]. We revealed only one

cytogenetic character which could support the basal position of Lasiopodomys in the tribe

Arvicolini: karyotypes of all studied species of the genus Lasiopodomys have two fragments of

MAG1 (see S1 Table), with the exception of the L. mandarinus karyotype in which three seg-

ments of MAG1 (LMAN2) were detected as a result of a secondary inversion. Thus, the two-

segment state of MAG1 links the genus Lasiopodomys and tribe Arvicolini, whereas the possi-

ble presence of three segments homologous to MAG1 was shown to be part of AAK [25].

The presumptive ancestral karyotype of the whole genus Lasiopodomys (LAK) could be

formed from AMiK by three Robertsonian fusions (Fig 4 and S1 Fig). According to phyloge-

netic estimates the basal radiation of genera Dicrostonyx, Prometheomys, Ondatra and tribe

Lemmini took place 6.5, 6.8, 7.7 and 7.2 MYA, respectively, and the divergence time of Arvico-

linae/Cricetinae was calculated to be 18.1 MYA [3]. Based on these data we can assume that

AMiK was formed about 3 MYA and LAK–about 2.4 MYA. So the rate of chromosomal

Karyotype Evolution in Lasiopodomys

PLOS ONE | DOI:10.1371/journal.pone.0167653 December 9, 2016 9 / 15

rearrangements in the branch leading from AAK to LAK is about one rearrangement per 3

million years (MY). However, the genus Lasiopodomys demonstrates an increased rate of chro-

mosomal rearrangements in comparison with the most other rodents: the karyotype of L. gre-galis evolved from the presumptive LAK by one fission and seven fusions, roughly four

chromosomal rearrangements per MY.

The putative common ancestral karyotype for the subgenus Lasiopodomys (sLAK) could

have been formed from LAK by five fissions and four fusions (Fig 4 and S1 Fig); the karyotype

of L. brandtii could be produced from sLAK mainly by Robertsonian fusions (9 fusions) and

one fission. Interestingly, the karyotype of L. mandarinus was shaped by a number of complex

Fig 4. Karyotype evolution pathways in three Lasiopodomys species. Tree topology is based on the molecular phylogeny of Arvicolinae species

presented by [3]. AAK–ancestral Arvicolinae karyotype, AMiK–ancestral karyotype of the tribe Arvicolini, LAK–ancestral karyotype of the genus

Lasiopodomys, sLAK–ancestral karyotype of the subgenus Lasiopodomys. Chromosome numbers are indicated in AAK, LAK, and sLAK segments. *–see

Discussion.

doi:10.1371/journal.pone.0167653.g004

Karyotype Evolution in Lasiopodomys

PLOS ONE | DOI:10.1371/journal.pone.0167653 December 9, 2016 10 / 15

fissions, fusions and inversions (Fig 4 and S2 Fig). In total, both species have undergone

around 20 chromosomal rearrangements in 2.4 MYA [3]. So, the rate of chromosomal rear-

rangements was increased in subgenus Lasiopodomys up to eight chromosomal rearrange-

ments per MY.

So the rate of chromosomal exchanges steadily increased during formation and radiation of

the Lasiopodomys species complex. Interestingly, we observed that the number of ribosomal

DNA clusters has the opposite trend and is much lower in currently evolving L. mandarinus(three) than in constant karyotypes of L. brandtii and L. gregalis (six) (Figs 1 and 3E–3G). Pre-

viously, it was hypothesized using the ground vole as an example that the “primitive” (ances-

tral) karyotype is defined by the high number of NORs, whereas species with extensively

rearranged karyotypes have a lower number of NORs [51]. Although we have previously

shown the hypothesis of multiple NORs as a characteristic of the primitive state this does not

hold in other species of voles [25]. Perhaps in this case, the few rDNA clusters in the Mandarin

vole was a consequence of the extensive chromosomal rearrangements that shaped the species

karyotype.

Interestingly, the increased rate of chromosome reshuffling in Lasiopodomys is not the only

sign of fast evolutionary genomic changes. An extremely high mutation rate in the mitochon-

drial cytochrome b gene was shown in L. gregalis during a previous species-wide phylogeo-

graphic study. The estimated rate is an order of magnitude higher than previous estimates for

Microtus species. A high genetic diversity was revealed both among and within the narrow-

headed vole mtDNA lineages [3,10].

Conclusion

Our molecular cytogenetic analyses have discovered chromosomal associations shared by L.

gregalis with L. mandarinus and L. brandtii, which unite the three species and support the

monophyly of Lasiopodomys. The karyotypes of Lasiopodomys have evolved through a compli-

cated chain of reshuffling events involving Robertsonian fusions, chromosomal fissions, inver-

sions and heterochromatin expansion. The findings also provide strong support for the

previously suggested subgeneric division of genus Lasiopodomys: the subgenus Stenocranius is

separated from subgenus Lasiopodomys by eight rearrangements that occurred within a short

evolutionary period, less than 1.8 MY [3,10]. The fast tempo of chromosome evolution

involved not only autosomes, but also sex chromosomes, forming an unusual complex of sex

chromosomes in at least one specimen of the Mandarin vole, with an elaborate combination of

expanded heterochromatin and autosomal/sex chromosomal rearrangements. We provide

molecular cytogenetic evidence that validates the hypothesis of autosomal translocation to sex

chromosomes [17] and explains the heteromorphism of the X chromosomes in L. mandarinus.We further hypothesize that the L. mandarinus karyotype originated over a short evolutionary

period (less than 1.8 MY) by extensive genomic rearrangements: 10 fusions/fissions, rDNA

location, ITS, heterochromatin expansion, and sex chromosomes rearrangements. We assume

that these processes might be still ongoing in the current populations. It remains to be

answered why karyotypes of L. gregalis and L. brandti that were formed as result of fast chro-

mosome evolution do not exhibit signs of cytogenetic variation in current populations. Thus,

cytogenetic studies of larger number of individuals from the Lasiopodomys genus could pro-

vide more clues as to the role of chromosome rearrangement in complex speciation events.

Supporting Information

S1 Fig. A hypothetical path of formation of the presumed ancestor karyotype of the genus

Lasiopodomys (LAK) from the ancestral karyotype of the tribe Arvicolini (AMiK) [11] and

Karyotype Evolution in Lasiopodomys

PLOS ONE | DOI:10.1371/journal.pone.0167653 December 9, 2016 11 / 15

a hypothetical path of formation of the presumed ancestral karyotype of L. mandarinusand L. brandtii (sLAK) from LAK (ancestral Lasiopodomys karyotype). Single chromosomes

of M. agrestis represented by one element in AAK and LAK are shown in blue. The chromo-

somes represented by two elements are marked by pairs of various colors. Numbers along the

segments correspond to chromosome numbers of M. agrestis. Plus signs indicate chromosome

fusions. �–see Discussion.

(TIF)

S2 Fig. A hypothetical path of evolutionary rearrangements of key chromosomes leading

to L. mandarinus karyotype formation from sLAK. Numbers along the chromosomes corre-

spond to chromosome numbers of M. agrestis. Minus signs indicate chromosome fissions,

plus signs indicate chromosome fusions. �–see Discussion.

(TIF)

S1 Table. Number of chromosomal segments painted by isolated M. agrestis and D. torqua-tus chromosomes in individual voles of L. mandarinus, L. brandtii and L. gregalis studied

here.

(DOC)

Acknowledgments

We are highly obliged to L.L. Voita (ZIN RAN) for assistance in obtaining the materials.

Author Contributions

Conceptualization: ASG FY.

Formal analysis: OLG SAR VAT.

Funding acquisition: ASG FNG SAR.

Investigation: MAFS NAL NAS OLG PCMOB SAR VAT.

Project administration: ASG SAR.

Resources: AVS FNG JMK.

Supervision: ASG.

Visualization: OLG SAR.

Writing – original draft: OLG SAR.

Writing – review & editing: FY MAFS PCMOB PLP VAT.

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